Our long term goal in experiments proposed here is to identify the mechanism(s) responsible for the down-regulation of the slow skeletal troponin I (ssTnI) isoform, which occurs during post-natal maturation of the heart. Preliminary data underpin our hypothesis that the microRNA, miR-208a, is involved in silencing the cardiac expression of ssTnI after birth. Our experiments continue research supported by this proposal that demonstrated that expression of ssTnI in the adult heart of transgenic mice protects the myocardium against common stresses including familial dilated cardiomyopathy (DCM). Preliminary data demonstrate: i) increased expression of miR-208a with muscle specific miRs, miR-1 and miR-133a in developing cardiomyocytes, and ii) knockdown of miR-208a in cardiomyocytes by a 208a-antagomiR induces ssTnI expression. Our objectives are as follows: Aim #1 To determine mechanism(s) of ssTnI and miR-208a mediated regulation by (a) investigating interplay of miR-208a with thyroid hormone mediated down regulation of ssTnI, (b) analyzing the role of miR-208a in conjunction with other co-regulated miRNAs in ssTnI gene regulation, (c) investigating transcriptional mechanisms of ssTnI gene regulation, and (d) evaluating ssTnI as a direct target of miRNA- mediated regulation. Aim #2: To determine the relative impact of miR-208a KO on modifications of thin filament proteins, on myofilament response to Ca, on myocyte mechanics and Ca, and on in situ cardiac function. Aim #3. To determine whether reduced expression of miR-208a improves cardiac function in dilated cardiomyopathy (DCM) associated with an alpha-tropomyosin mutation. Our approach to Aim #1 includes i) analysis of expression of ssTnI in neonatal cardiac myocytes by gain and loss of function approaches to dissect the relative significance of thyroid related signaling in the repression of ssTnI; ii) determination the relative role of miR-208a as a repressor of ssTnI expression employing co-induction with other miRs; iii) identification of transcription factors targeted by miR-208a and determination of their DNA binding and expression; and iv) identification of targets of miR-208a in ssTnI 3'UTR. Our approaches to Aims #2 include studies comparing miR208a-KO mice to controls to determine the functional impact of altered myofilament properties as reflected in isoform population, post-translational modifications, pCa-force relations and cross- bridge kinetics. Findings from studies of skinned myocytes are integrated into myocardial function by studies of Ca2+ transients and mechanics at the level of cardiac myocytes and of dynamics of in situ beating hearts responding to stresses including those dominated by altered thin filament properties. In Aim #3 our approach is to determine whether cross-breeding a familial DCM mouse model linked to a tropomyosin mutation with the miR208a-KO mouse is able to rescue the DCM phenotype. Experiments proposed here fill a significant gap in our understanding of the control of expression of the isoform population of myocardial TnI, an essential thin filament protein, and test a significant therapeutic approach to cardiac disorders.